Biological self-protection inspired engineering of nanomaterials to construct a robust bio-nano system for environmental applications

Nanomaterials can empower microbial-based chemical production or pollutant removal, e.g., nano zero-valent iron (nZVI) as an electron source to enhance microbial reducing pollutants. Constructing bio-nano interfaces is critical for bio-nano system operation, but low interfacial compatibility due to nanotoxicity challenges the system performance. Inspired by microorganisms’ resistance to nanotoxicity by secreting extracellular polymeric substances (EPS), which can act as electron shuttling media, we design a highly compatible bio-nano interface by modifying nZVI with EPS, markedly improving the performance of a bio-nano system consisting of nZVI and bacteria. EPS modification reduced membrane damage and oxidative stress induced by nZVI. Moreover, EPS alleviated nZVI agglomeration and probably reduced bacterial rejection of nZVI by wrapping camouflage, contributing to the bio-nano interface formation, thereby facilitating nZVI to provide electrons for bacterial reducing pollutant via membrane-anchoring cytochrome c. This work provides a strategy for designing a highly biocompatible interface to construct robust and efficient bio-nano systems for environmental implication.

the incubation time.
NADH level.The NADH content was determined through the WST-8 reaction by NADH/NAD + Assay Kit with WST-8 (Beyotime Biotechnology).The bacteria in bionano systems were centrifuged at 6000 rpm at 4 o C for 5 min, suspended in a buffer solution, and then the NADH extract was added to lyse the cells.The lysed cells were centrifuged at 12000 rpm at 4 o C for 10 min, and the supernatants were taken into 96well plate after heating in a water bath.Color developing agent was added to the plate, and the absorbance was measured at 450 nm for determination.

Transcriptomic analysis.
Total RNA was isolated and extracted, and the total amount and integrity of RNA was assessed using an RNA Nano 6000 assay kit (Bioanalyzer 2100, California, America).Probes were used to eliminate rRNA in order to purify the total RNA and construct sequencing libraries.The reference genome and gene model annotation files (S. oneidensis MR-1) were obtained from NCBI [https://ftp.ncbi.nlm.nih.gov/genomes/all/GCA/000/012/525/GCA_000012525.1_ASM1252v1/] for read mapping using Bowtie2-2.2.3.DESeq R package (1.18.0) was used to analyze the differential expression of the control group and the experiment group.

Two-chamber galvanic cell experiment.
A proton exchange membrane separated the two cell chambers, with nZVIbio loaded carbon paper as the anode and carbon felt as the cathode.Titanium wire connected the electrodes.Both the anode and cathode chambers were filled with 100 mL of mineral medium without sodium lactate, and the cathode chamber additionally contained NBS (50 mg/L).The washed bacterial suspension was added to the cathode solution to achieve an initial OD600 of 0.2.During the reaction process, the two-chamber galvanic cell system was connected to a data collector to monitor the voltage difference between the cathode and anode.Periodic sampling of the cathode solution was performed to determine NBS and ABS concentration.The carbon paper and carbon felt were collected for SEM and EDS mapping after the reaction.

Characterization of nanomaterials and bio-nano systems.
The particle size distribution and zeta potential of nZVIbio and nZVI were measured using Malvern instrument (Zeta sizer Nano series, Malvern).The specific surface area was measured by a Surface Area and Porosity Analyzer (MicromeriticsTristar II 3020, USA).To determine the hydrophilicity of each material, equal mass powder (0.1 g) was compressed using a tablet press and water contact angles were measured with a contact angle goniometer (JC000D1, Powereach).Electrochemical characterizations, including open circuit potential (OCP) and Tafel scans, were measured in an oxygen-free 50 mM Na2SO4 solution using nZVIbio or nZVI loaded glassy carbon as the working electrode, platinum wire as the counter electrode, and Ag/AgCl (saturated KCl) as the reference electrode.The nZVIbio or nZVI loaded working electrode was prepared by a two-step tablet pressing method (26).After recording OCP for 10 min, Tafel scanning was performed with the three electrode system to record free corrosion potentials.In preparation for differential pulse voltammetry (DPV), 250 μL of EPS solution was combined with 250 μL of isopropyl alcohol and 10 μL of Nafion aqueous solution.This mixture was drop-casted onto a glassy carbon electrode and allowed to dry before being immersed in the electrolyte for testing (21,40).The parameters of DPV were as follows: Ei = -0.6V; Ef = 0.4 V; amplitude, 50 mV; pulse width, 300 ms; and potential increment, 5 mV.The crystal structure and chemical composition of nZVIbio or nZVI before and after reaction were characterized by X-ray diffractometer (TTR III, Japan) and X-ray photoelectron spectroscopy (Thermo Scientific ESCALAB 250X, USA).

SPR analysis
Binding experiments of nano-iron particles on bacteria were performed with SPR device (BI-4500, Biosensing Instrument Co., USA).Each nanoparticles solution was injected at a flow rate of 25 μL/min, and with a contact time of 400 s.All measurements were carried out at 25 o C. The concentration of nanoparticles was at a batch experimental dose of 50 mg/L.The binding process of nanoparticles on bacteria could be described by where ∆ is the change of the SPR signal after deducting the background,   is the SPR signal of binding equilibrium and   is the apparent rate constant (1/s).

Nanomaterials and bio-nano systems preparation protocols for TEM.
A suspension of bacteria and nanomaterials was briefly centrifuged and fixed with precooled 2.5% glutaraldehyde at 4 o C for 10 min.Subsequently, another centrifugation at 6000 rpm for 5 min was performed after decanting the culture solution.After removing the supernatant, the bacteria were fixed overnight at 4 o C in a fresh fixing solution (5% glutaraldehyde and 4% paraformaldehyde).The fixing solution in the sample was sucked out and transferred to the recovery bottle.Subsequently, 0.1 M PB buffer (pH = 7.2) was added and rinsed three times at room temperature.Then the solution in the sample was sucked out and fixed at room temperature with 2% osmium tetroxide.The waste liquid was recycled, and the sample was rinsed three times with 0.1 M PB buffer (pH = 7.2) at room temperature.The washed samples were dehydrated with sequential treatment with 30, 50, 70, 80, 90, and 100% ethanol for 20 min.The samples were then infiltrated and embedded in Spurr's resin with propylene oxide (treatment with 1:1 and 1:3 of propylene oxide/Spurr's resin mixtures for 1 h and 3 h, and 100% Spurr's resin for 24 h).Finally, the polymerized blocks were placed on the top layer of the oven at 70 o C and polymerized for over 8 h.The polymerized blocks were sectioned using an ultra-microtome (Leica UC7, Germany) and observed via a transmission electron microscopy (H-7650, Japan).

Bacteria and bio-nano systems preparation protocols for SEM.
Cells before and after treatment with nZVIbio or nZVI were collected via centrifugation.The obtained cells underwent three washes with sterile 0.85% NaCl and were fixed overnight with 2.5% glutaraldehyde at room temperature.Next, the cells were collected by centrifugation and washed three times with 0.85% NaCl.The washed samples were dehydrated with sequential treatment with 30, 50, 70, 80, 90, and 100% ethanol for 20 min.The samples were sputtered with Au and characterized by a scanning electron microscope (GeminiSEM 500, USA).

Liquid and gas chromatography analysis.
The concentrations of NBS and ABS were determined by a high performance liquid chromatography (Model 1260, Agilent) with a C18 column (Agient Technologies, 5 μm, 4.6 × 250 mm).The mobile phase comprised a mixed solution of tetraethylammonium bromide (1 g/L) and methanol, with a ratio of 40:60 (v/v) and a flow rate of 0.5 mL/min.Detection of signals was set at 254 nm.The concentration of sodium lactate was determined by a high performance liquid chromatography (Model 1260, Agilent) with a hydrogen column (Hi-Piex H, 300 × 7.7 mm).Hydrogen concentration in the headspace of a 60 mL serum vial was detected using a gas chromatography (SP-6890, Lunan Corp).

Determination of NO3 -concentration.
The concentration of NO3 -was determined by a spectrometer (UV2600, Shimadzu).Briefly, 400 μL sample was added to a 5 mL centrifuge tube with 1.6 mL deionized water, followed by 16 μL of 0.2% sulfamic acid and 10 μL of 1 M HCl.The solution and standard product were spectrophotometrically analyzed for absorbance at 220 nm and 275 nm after thorough mixing.The corrected nitrate absorbance was obtained by subtracting twice the absorbance at 275 nm from the absorbance at 220 nm.Fig. S5.TEM images of bio-nano systems.TEM images of (A) bacteria, (B) bio-nZVI system and (C) bio-nZVIbio system at the same magnification.TEM images of (D) bio-nZVI system and (E) bio-nZVIbio system at the higher magnification.